We assessed the microbial causes of neonatal conjunctivitis by comparing 55 infants with purulent conjunctivitis and 60 healthy control infants. A mean of greater than 5 leukocytes per 1000X microscopic field was seen in Gram-stained smears obtained from the more inflamed eye in 77% of 30 untreated patients but none of 57 controls. Pathogens isolated more often from untreated patients than from controls included Haemophilus spp. (17% vs 2%, P = 0.01), Staphylococcus aureus (17% vs 2%, P = 0.01), Chlamydia trachomatis (14% vs 0%, P = 0.01), enterococci (8% vs 0%, P = 0.05), and Streptococcus pneumoniae (11% vs 2%, P = 0.06). One or more of these pathogens were isolated from the conjunctivae in 58% of patients and 5% of controls (P less than 0.001). Bacterial morphology seen on smear correlated with the pathogens cultured. Isolation of Haemophilus spp. or S. pneumoniae was associated with dacryostenosis. We conclude that several microbial pathogens are implicated in neonatal conjunctivitis. These organisms have differing susceptibilities to antimicrobial agents, so culture and sensitivity testing are required as a guide to therapy.